Fundam. appl. NemalOl., 1996, 19 (4), 369-374
Chromosome number of the potato cyst
nematode Globodera rostochiensis
Jeroen N. A. M.
Ellen L.J. G. VAN ENCKEVORT, Laas P. PIJNACKER*,
Johannes HELDER, Fred J. GOMMERS and Jaap BAKKER
ROUPPE VAN DER VOORT,
Departmenl of Nematology, Wageningen Agricultural University, Binnenhaven JO, 6709 PD, Wageningen, The Netherlands.
Accepted for publication 17 July 1995.
Sununary - The chromosome number of Globodera roslOchiensis was deterrIÙned by investigating : i) bivalent configurations at
metaphase l, ii) meioricalJy reduced cells during oogenesis and iit) somatic rIÙtosis at early embryonic cleavage divisions. A diploid
chromosome number of 2n = 18 was found. Chromosome numbers were readily deterrIÙned in polar bodies using the fluorochrome
Hoechst 33258. An irnproved squashing method enabled observations at early cleavage divisions.
Résumé - Nombre de chromosomes du néTnatode à kyste de la pomme de terre Globodera rostochiensis - Le nombre
de chromosomes de Globodera TOslOchiensis a été déterminé par l'analyse i) de leurs configurations bivalentes au stade métaphase l, ii)
de cellules au stade de réduction méiotique pendant l'oogenèse, et iii) de cellules en rIÙtose somatique au cours de divisions
embryonnaires prématurées. Le nombre de chromosomes diploïde est 2 n = 18. L'utilisation du fluorochrome Hoechst 33258 a
facilité la détennination du nombre de chromosomes dans les globules polaires. Les prerIÙères divisions ont été observées en utilisant
une méthode d'écrasement améliorée.
Key-words : Chromosome number, embryogenesis, Globodera, holocentric behaviour, oogenesis.
The potata cyst nematode species Globodera rostochiensis (Woll.) Skarbilovich and G. pallida Stone are
obligate plant parasites which are serious pests in potato
crops. Populations of both species are classified into
pathot:ypes as defmed by their ability or inability to reproduce on a standard set of potato clones (Kort et al.,
1977). One of these clones, Solanum tuberosum ssp. andigena CPC 1673, harboring the H1-resistance gene, is
used to define the G. rostochiensis pathotypes RoI' and
R0 4 . A gene-for-gene relationship was demonstrated for
the interaction berween G. rostochiensis and the H [-resistance gene. Virwence is inherited at a single locus and
is recessive to avirulence Oanssen et al., 1991). This
gene-for-gene interaction is used as a model to unravel
the molecwar basis ofvirtùence in the potato cyst nematode G. rostochiensis. Currently we are tracing molecular
markers linked ta the avinùence gene in backcross populations (Rouppe van der Voort et al., 1994). Progress is
restrained by serious gaps in the knowledge in the genetic constitution of this bisexually-reproducing cyst nematode species.
Data on the chromosome number of potato cyst nematodes are scarce and conflicting. Riley and Chapman
(1957) observed nine pairs of chromosomes at first
metaphase ofmeiosis (2n = 18) in Heterodera rostochiensis. It is noted that until 1970 Oones et al., 1970; Stone,
1973) both potata cyst nematode species were consid-
* Address: Deparlment of Genelies,
ered pathotypes of a single species, H. rostochiensis. Variations in the basic chromosome number of H. rostochiensis were reported by Cotten (1959,1960). He observed
diploid chromosome numbers of20 and 22. OccasionalIy, 19, 23 and 24 chromosomes were found. Although
nearly indistinguishable morphologically, the rwo potato
cyst nematode species are discrirninated by 70 % of their
polypeptides as resolved by rwo-dimensional gel e1ectrophoresis (Bakker & Bouwrnan-Smits, 1988). These data
indicate that G. rostochiensis and G. pallida diverged millions of years ago. Hence, the discrepancy in chromosome numbers as reported by the afore-mentioned papers can be explained by the possibility that a mixture of
different species were analysed. Recently, the chromosome complement of one of the rwo species, G. pallida,
was determined to be 2n = 18 although considerable
variation was observed (Grisi et al., 1995). The authors
stated that part of this variation cowd be due to observational errors.
In this report, the chromosome number of G. rostochiensis was investigated. Using the f1uorochrome
Hoechst 33258 and an irnproved squashing method, the
chromosome number was established by : i) observation
of bivalent configurations at metaphase 1, il) the reduction process of the diploid complement during oogenesis and iù) determination of the re-established somatic
number at early embryonic cleavage divisions.
Cenlre of Biological Science, Universily of Groningen, Kerklaan 30, 9551 NN, Haren, The Nelher-
lands.
ISSN 1164-5571196/04
$ 4.00 / © Gauthier-Villars - ORSTOM
369
J. N.
A. M. Rouppe van der Voort et al.
Materials and lYlethods
MICROSCOPY
POPULATIONS AND UNES
A Leitz Wetzlar Orthoplan microscope with a 100 x
oil immersion objective was used to examine the
Hoechst 33258 stained slides. The filter combination
BP 355-425 / LP 460 was used to visualize the nuclei.
Objects were photographed with a Kodak Ektachrome
P 800/1600 fùm adjusted at 400 ISO. The Feulgen
stained nuclei and eggs were visualized with a 100 x oil
immersion objective and photographed with Agfa Ortho
film (25 ISO). Chromosome lengths were determined in
Feulgen stained nuclei using a stage micrometer (Leitz
Wetzlar).
The G. TOstochiensis population" Mierenbos ", classified as Ro] was obtained by the Plant Protection Service,
Wageningen and multiplied in our laboratory. A virulent
(Ro ç 22) and an avirulent (Ro l -19) inbred [ine of G.
rostochiensis were derived from controlled single matings
and selected as described previously Qanssen et al.,
1990).
The G. rostochiensis lines were reared on the susceptible clone S. tuberosum ssp. tuberosum L. cv. Eigenheimer. Potato plants were inoculated with approximately
200 cysts and placed in a growth chamber at 18 oC and
16 h daylength. To harvest adult females, root balls were
inspected daily. Young females outside the roots were
collected from the potato roots with a small brush.
FIXATION
Collected females were immediately fixed in cold 3 : 1
(v/v) ethanol-acetic acid and stored at - 20 oC for at
least 5 days. This treatment caused increased permeability of the egg wall for cytological stains and facilitated squash preparations of eggs. Fixation was followed
by either Hoechst 33258 or Feulgen staining.
SQUASH PREPARATIONS AND HOECHST STAINING
Microscope slides were pre-treated in a 1 % gelatin
solution and subsequenùy air-dried. White females were
placed on the slides, the adhering fixative was removed
with tissue paper and a drop of 45 % (v/v) acetic acid
was added. Females were opened with fme needles to
release the body content. Gonads were squashed under
a cover-slip (24 x 50 mm) and the egg contents were
spread. Slides were dipped in liquid nitrogen and the
cover-slip was then removed using a razor blade. The
slides were air-dried, rinsed in 95 % (v/v) ethanol and
fixed overnight in 3 : 1 (v/v) ethanol-acetic acid. Before
staining, the slides were air-dried again and placed for
5 min in PBS (Sulston & Hodgkin, 1988). Preparations
were stained for 5 min in 1 f..l.g/rnl Hoechst 33258 (bisbenzimide, Sigma) dissolved in PBS. FinalJy, the slides
were rinsed in bidistilled water.
FEULGEN STAINING
After removing the adhering fixative, young females
were incubated in 5N HCI for 25 min at room temperature. This results in a hydrolysis of the purine-deoxyribose bonds. Reactive aldehydes were demonstrated by
Schiffs reagents (Feulgen & Rossenbeck, 1924). The
incubation rime was 3 h at 4 oc. Thereafter, females
were rinsed in tap water until the water remained clear.
Up to three srained females were placed on a slide,
water was removed and a drop of 45 % (v/v) acetic acid
was added. The females were opened with fme needles
and the body contents was smeared on the slide. A cover
slip was placed over it and pushed genùy.
370
Results
The potato cyst nematode, G. rostochiensis did not
show intraspecific variation in chromosome behaviour
du ring maturation divisions and embryogenesis among
the populations tested. The data presented here are representative for the Ro1-Mierenbos population and the
lines RO I -19 and Ros-22.
CHROMOSOME PAlRtNG AT METAPHASE
l
Chromosome pairing was clearly observed at diakinesis/metaphase 1. At this stage the nuclear membrane
disappeared. Nine bivalents of different sizes became
discernable (Fig. 1A). The chromosomes were rodshaped and lack visible constrictions. Each chromosome
consisted of two separated chromatids which were arranged side by side, apparenùy perpendicular to the
equatorial plane.
REDUCTION OF THE DIPLOlD COMPLEMENT DURING
OOGENESIS
The first maturation division resulted in two haploid
nuclei with a chromosome number of n = 9; the egg
nucleus and the first polar nucleus, both with condensed
chromosomes. The spindle was situated perpendicular
to the cell membrane and the first polar nucleus ended
near the periphery of the oocyte. The egg nucleus proceeded to anaphase II (Figs 1 B, 2 A) whereas the first
polar nucleus maintained its telophase l configuration
(Fig. 2 B). The chromatids separated by parallel disjunction during anaphase II, and no centromere was visible (Fig. 1 B). The second maturation division resulted
in the formation of a second polar body and the egg
pronucleus.
The amount of fluorescence reflected also the reduction of the diploid complement into the haploid condition (n = 9), which was accompanied with a change
from 4 C to 2 C in the first meiotic division and to 1 C in
the second division (Fig. lA, B, C). The sperm pronucleus, which was localized at one pole of the oocyte
(Fig. 2 C), moved towards the egg pronucleus and they
fused to form the zygote nucleus. Polyspermy within
one egg was never observed.
Pundam. appl. NemalOl.
Chromosome number in Giobodera rostochiensis
Fig. 1. Chromosomes and nuciei of Globodera rostochiensis Slained wilh lhe fluorochrome Hoechsl 33258. A: Melaphase 1 wilh nine
bivaœnts; B : Anaphase Il; C: Firsl cœavage division [Al bOlh sides of lhe cieavage plaie (cp) IWO somalie nuclei are discernible; lhe jim (l)
and lhe second polar body (lI) are indicaled},o D: Second cleavage division; E: Firsl polar nucleus, n = 9; F: Firsl polar nucœus, n = /0.
(Scaiebar= 10f.Lm.)
Vol. 19, n° 4 - 1996
371
J. N.
A. M. Rouppe van der Voorl et al.
Fig. 2. A single oocyte al lhree focal pwnes wùh A : Anaphase II; B : Firsl polar nucleus; C : Sperm pronucleus (FeuJgen srairùng; only the
objects marked with an arrow are Feulgen stained. Scale bar = 10 fLm).
SOMATIC CHROMOSOME NUMBER AT EARLY
EMBRYONIC CLEAVAGE DIV1SIONS
Early cleavage divisions showed the restored somatic
number of eighteen chromosomes (Fig. 3). These mitotic chromosomes were much more elongated than the
meiotic chromosomes and showed no primary consn1ctions. Both polar bodies remained visible during early
embryonic development on top of the yolk, close ta the
egg wall (Fig. 1 C). While the frrst polar body, still with
recognizable chromosomes persisted almost until completion of embryogenesis, the second polar body disintegrated soon after the frrst divisions. Different mitotic
phases within one embryo indicated that daughter cells
did not divide simultaneously from the first cleavage
division onwards (Fig. 1 D).
DETERMINATION OF CHROMOSOME NUMBER
Fig. 3. MÙOlic chromosomes al melaphase in a young embryo
slained wùh Feulgen. (The inset is a diagranunic representation.
Scale bar = IOfLm.)
The diploid chromosome number of G. roslOchiensis
was determined to be 2n = 18. Most observations of
chromosomes were made on haploid nuclei of the first
polar body (Fig. 1 E). The chromosomes of these nuclei
were free from cytoplasm and were most definite as
compared to other nuclear stages. Seven chromosomes
varying in length from 0.8 to 1.2 IJ..m could usually be
distinguished from a small chromosome (0.6 IJ..m in
length) and a large chromosome (1. 5 IJ..m in length). In
372
Fundam. appl. NemalOl.
Chromosome number in Globodera rostochiensis
only two out of the 200 polar nuclei examined, one extra
chromosome was observed (Fig. 1 F).
Discussion
Despite the extensive divergence at the molecular level between G. roslOchiensis and G. pallida (Bak.ker &
Bouwman-Smits, 1988; De Jong et al., 1989; Folkertsma et al., 1994) both species share a similar chromosome complement of 2n = 18. This chromosome number is analogous to karyotypes found in other cyst
nematades (genera Globodera, Heterodera), with the exception of H. betulae (Triantaphyllou, 1975). The karyotypic uniformity between Globodera species helps to
explain the capacity of these extensively diverged species ta produce interspecific hybrids. Matings between
G. roslOchiensis and G. pallida resulted in viable, although not fertile, second stage juveniles (Mugniéry,
1979; Mugniéry et al., 1992).
Variation in the chromosome number of G. rostochiensis was much lower than the variation reported by
Cotten (1959, 1960) for G. roslOchiensis and by Grisi et
al. (1995) for G. pallida. Only one percent of the polar
nuclei showed a deviant chromosome number. TriantaphyUou (1975) observed in a small number of females
of two H. schachtii populations an extra chromosome
which was transmitted to 50 % of the progeny of these
females. The extra chromosome divided normally during maturation divisions and was considered to be a
supernumerary chromosome. However, because of the
low frequency of an extra chromosome and because this
extra chromosome can be the result of non-disjunction
during anaphase II, it seems unlikely that supernumerary chromosomes are common in the G. rostochiensis
populations studied.
The use of fluorochrome Hoechst 33258 facilitated
the counting of potato cyst nematode chromosomes.
Hoechst 33258 interacts specifically with DNA. Its fluorescence increases the microscopic resolution and the
chromosomes were mosùy observed within one plane.
The first polar nuclei are especially suitable for karyotype determination. The absence of cytoplasmic counterstaining and the persistence of polar nuclei during
embryogenesis enable unequivocal determinations of
the haploid chromosome numbers. This could be used
for karyotype analyses in other nematode species.
For a number of nematode species including Caenorhabditis elegans, Meloidogyne hapla, Parascal1's equorum,
and P. univalens, chromosomes lacking a localized centromere are found (White, 1973; Goldstein & Triantaphyllou, 1980; Albertson & Thomson, 1982; Pimpinel!i & Goday, 1989). Although by no means proven,
the side by side arrangement of the chromatids at metaphase l, the paral!el segregation at anaphase II, and the
absence of distinct centromeric constrictions suggest a
holocentric nature of Globodera chromosomes.
Vol. 19, n° 4 - 1996
It has been argued that the holocentric nature of nematode chromosomes may have been evolved ta prevent cel! death due ta chromosome breakage (Pimpinelli
& Goday, 1989). Characteristic for nematode genomes
is their mosaic embryonic development in which each
cell passes through a determined number of mitatic divisions and cannot be replaced by another cell. Breakages
in monocentric chromosomes would be lethal to the cell
and would thus affect the whole embryo. Holocentric
chromosomes are characterized by a diffuse centromere
which extends along the length of the chromosome.
Fragmented holocentric chromosomes may still conta in
a spindle attachment site and remain through cell division. Holocentric chromosomes may provide nematodes a strategy ta circumvent lethal effects of chromosome fragmentation.
Finally, it is concluded that the chromosome number
of the G. rostochiensis populations studied is 2n = 18 and
constant. From strict bivalent pairing, observed in metaphase l, Mendelian segregation of maternai and paternal
alleles is expected. This is of importance for a genetic
analysis of the potata cyst nematode with molecular
markers.
Acknowledgements
We wou Id like to acknowledge Dr. J. H. de Jong of the
Departmenr of Genetics, Wageningen Agriculrural University
and Ms M. Ferwerda of the Department of Genetics, University of Groningen. The research is supported by a grant of
the Programme committee on Agricu1rura! Research of the
Netherlands (PcLB) and the EC-grant AIR3 CT 92.0062.
References
ALBERTSON, D. G. & THOMSON, J. N. (1982). The kinetochores of Caenorhabditis elegans. Chromosoma, 86 : 409428
BAKKER, J. & BOUWMANN-S'\1lTS, L. (1988). Contrasting
rates of protein and morphological evolution in cyst nematode species. PhYlOpathology, 78 : 900-904.
CO'l''l'EN, J. (1959). Chromosome number of the potato root
eelworm, Heterodera l'OslOchiensis Wollenweber. Nature,
183: 128.
COTTEN, J. (1960). Observations on the cytology of the potato-root eelworm, Hetel'Odera roslOchiensis Wollenweber. Nemawlogica, Suppl. II .' 123 : 126.
DE JONG, A. ]., BAKKER, ]., Roos, M. & GOMMERS, F. J.
(1989). Repetitive DNA and hybridization patterns demonstrate extensive variability between the sibling species Globodera l'OslOchiensis and G. pallida. Parasitology, 99: 133138.
FEULGEN, R. & RossENBEcK, H. (1924). Mikroskopischchemischer Nachweis einen Nucleinsaure von Typus der
Thymonuclinsaure und die darauf berhende elektive Farbung von Zellkernen in microskopischen Preparaten.
Hoppe-Seyler's Z. physiol. Chem., 135 : 203-248.
373
J. N.
A. M. Rouppe van der Voorl et al
FOLKERTSMA, R. T., ROUPPE YAN DER VOORT,}. N. A. M.,
YAN GENT-PELZER, M. P. E., DE GROOT, K. E., VAN DEN
Bos, W. J., SCHOTS, A., BAKKER, }. & GOlVLVlERS, F. }.
(1994). Inter-and intraspecific variation berween populations of Gwbodera l'oslOchiensis and G. pallida revealed by
random amplified polymorphie DNA. PhYlOpalhowgy, 84 :
807-811.
GOLDSTElN, P. & TRlANTAPHYLLOU, A. C. (1980). The uJtrastrucrure of sperm development in the plant-parasitic nemarode Meloidogyne hapla. J. Ullraslr. Res., 71: 143-153.
GRlSI, E., BURROWS, P. R., PERRY, R. N. & HOJ\1JNICK, W.
M. (1995). The genome size and chromosome complement
of the potaro cyst nematode Gwbodera pallida. Fundam. appl.
NemalOl., 18: 67-70.
JANSSEN, R., BAKKER}. & GOMNlERS, F.}. (1990). Selection
of virulent and aviruJent lines of Gwbodera roslOchiensis for
the HI resisrance gene in Solanurn tuberosum spp. andigena
CPC 1673. Revue NérnalOl., 13: 265-268
JANSSEN, R., BAKKER}. & GOlvIMERS, F.}. (1991). Mendelian
proof for a gene-for-gene relationship between virulence of
Globodera roslOchiensis and the HI resistance gene in Solanum tuberosurn spp. andigena CPC 1673. Revue Némawl.,
14: 207-211.
JONES, F. G. W., CARPENTER, J. M., PARROTT, D. M.,
STONE, A. R. & TRUDGlLL, D. L. (1970). Potato cyst nema rodes : one species or two? Nalure, 227 : 83-84.
KORT, ]., Ross, H., RUMPENHORST, H. J. & STONE, A. R.
(1977). An international scheme for identifying and classifying pathorypes of potato cyst nematodes Globodera rost.ochiensis and G. pallida. NemalOwgica, 23 : 333-339.
MUGNIÉRY, D. (1979). Hybridation entre Globodera rost.ochiensis (WoUenweber) et Gwbodera pallida (Stone). Revue
NérnalOl., 2 : 153-159.
374
MUGNIÉRY, D., BOSSIS, M. & PIERRE,}. (1992). Hybridations
entre Globodera rOSlOchiensis (Wollenweber), G. pallida
(Stone), G. virginiae (Miller & Gray), G. solanacearurn
(Miller & Gray) et G. "mexicana" (Campos-Vela). Description et devenir des hybrides. Fundarn. appl. NemalOl.,
15 : 375-382
PIMPlNELLl, S. & GODAY, C. (1989). Unusual kinetochores
and chromatin diminution in Parascaris. Trends in Genelics,
5: 310-315.
RILEY, R. & CHAPMAN, V. (1957). Chromosomes of the potato root eelworm. Nature, 180-662.
ROUPPE VAN DER VOORT, }. N. A. 1\1.., ROOSlEN, }. YAN
ZANDVOORT, P. 1\1.., FOLKERTSMA, R. T., YAN ENCKEYORT, E. L.}. G.,fANSSEN, R., GOMMERS, F.}. & BAKKER,
J. (1994). Linkage mapping in potato cyst nematodes. In :
Lamberti, F., De Giorgi, C. & McK. Bird, D. (Eds). Advances in molecular plant nernalOlogy. New York, USA, Plenum Press: 57-63.
STONE, A. R. (1973). Heterodera pallida n. sp. (Nematoda:
Heleroderidae), a second species.of the potaro cyst nemarode.
NemalOwgica, 18 (1972) : 591-606.
SULSTON, J. & HODGKIN,}. (1988). Methods. In: Wood, W.
B. (Ed.). The nemalOde Caenorhabditis elegans. Cold Spring
Harbor Laborarory : 587-606.
TRIANTAPI-lYLLOU, A. C. (1975). Oogenesis and the chromosomes of t:welve bisexual species of Het.erodera (Nematoda :
Heteroderidae). NemalOlogica, 7 : 34-40.
WHITE, M.}. D. (1973). Animal cYlOlogy and evolution. Cambridge, UK, Cambridge Universiry Press: 14-18, v + 961 p.
Fundarn. appl. Nernawl.